1. Using molecular modeling, CD and NMR spectroscopy.[unreadable] The three-dimensional structures of NK-2 class homeodomain proteins, the human NKX 2-1, NKX 2-5, and NKX 3-1 proteins, have been investigated by Dr. Gruschus in the free state, bound to DNA, and in ternary complexes with associated transcription factors. These proteins orchestrate organogenesis during embryonic development, and maintain organ cells in their differentiated state in adults, suppressing carcinogenesis. Recently in our laboratories, stabilizing and destabilizing interactions have been discovered between the homeodomain and motifs in the N and C-terminal flexible random coil regions. These motif interactions appear to be modulated by phosphorylation. The stuctural mechanisms by which these interactions stabilize and destabilize the homeodomain are our current focus. Additional results tie homeodomain stability with protein turnover in the cell. Thus, these N and C-terminal motifs and their interacting kinases could be targets for therapies aimed at modulating homeodomain protein levels. In particular, increasing NKX 3-1 stability is an important therapeutic goal since lowered NKX 3-1 protein levels have been implicated in prostate tumorigenesis.[unreadable] [unreadable] 2. Electron Microscopy and Tomography Image Processing.[unreadable] The EMAP module in CHARMM has been developed by Dr. Wu and is becoming a very useful tool in electron microscopy structure determination. This module employs the Grid-Threading Monte Carlo method to search for the best fitting structure and is highly efficient and reduces the fitting time by one or two orders of magnitude as compared with other software available in this field. [unreadable] [unreadable] 2.1. Structure study through single-particle cryoelectron microscopy.[unreadable] The pyruvate dehydrogenase (PDH)2 multienzyme complexes play a central role in cellular metabolism,catalyzing the oxidative decarboxy-lation of pyruvate to acetyl CoA,to link glycolysis and the tricarboxylic acid cycle. Here,we extend our previous studies with the report of the architecture of a 1:1 stoichiometric complex containing 60 copies each of the E2 and E3 enzymes.We present a cryoelectron microscopic analysis of the reconstructed three-dimensional structure of the purified E2E3 complex and use automated docking methods to interpret the density map in terms of the probable localization of the E2 and E3 molecules. Our findings show that the arrangement of E1 and E3 molecules in the outer shell of the pyruvate dehydrogenase complex are remarkably similar and indicate that the design of the annular gap allows the lipoyl domain to have access to the active sites of E1,E2,and E3 enzymes from within the annular gap.[unreadable] [unreadable] 2.2 Structure determination through single-particle electron tomography [unreadable] Single-particle electron tomography can be used to obtain 3D volume data of molecular systems without averaging many particles. This technology has the potential to study multiple conformational states of single particles. High noise due to limited sampling and distorsion caused by missing widges are two difficulties for this technology. Normal fitting methods often fail when dealing with such a high noisy data. The core-weighted fitting method we developed and implemented into CHARMM can tolerate such high noise and produce reasonable fitting results. We are applying the core-weighted fitting method to derive complex structures from single-particle electron tomography data. PDH complexes based on icosahedral symmetry are among the largest cellular machines and are a good candidate for single-particle electron tomography study. With the tomography image, we successfully docked the E2CD core and E1 domains and obtained complexes that agree with the results from cro-EM studies. These results validate the experiment techniques as well as our fitting methods for tomography data.[unreadable] Another ongoing project is to determine the structure of the Env spikes of HIV. The Env spikes of HIV-1 are composed of surface envelope glycoprotein (gp120SU) and transmembrane envelope glycoprotein (gp41TM) subunits, which, in their native configuration, elicit partially effective humoral immune responses and represent obvious vaccine targets for viral prophylactics. The Env spikes are thought to be trimeric and structure-based models have been proposed. However, despite intensive efforts, the arrangement and orientation of the loop-deleted gp120 core atomic structures within a native Env spike and their association with the gp41 subunits have remained largely speculative. Recently, Dr. Subramaniams lab obtained electron tomograms of the Env spikes on intact, chemically fixed SIV and HIV-1. We are now applying our image processing and fitting methods to reveal the distribution pattern of Env spikes on individual virions and to generate a three-dimensional (3D) model of the SIV Env spike. [unreadable] [unreadable] 2.3 Structure determination through of Chemotaxis receptor assemblies [unreadable] The image processing methods developed in our lab has been successfully applied in determination of the three-dimensional structure of a chemotoaxis receptor in E-Coli cell membrane by use of electron tomography and averaging of segmented volume densities from electron tomogram. This work bridges the gap between crystallography and cryoelectron tomography of whole cells and makes a major advance in understanding of bacterial chemotaxis. Although atomic structures of both periplasmic and crytoplasmic parts of bacterial chemoreceptors have been determined, the integral structure of the entire membrane-embedded molecules remains unresolved, as does receptor arrangement in the higher-order oligomeric complexes. Our results confirm previously suggested trimer-of-dimers arrangement of receptors. In addition, we reveals two conformational states of this signal transduction machinery, providing direct structural information in the study of signal transduction mechanism.[unreadable] [unreadable] 3. Protein-protein docking with Map Objects. [unreadable] We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Particularly, we implemented the core-weighted correlation functions to effectively recognize correct fit of component maps in complex maps, and the grid-threading Monte Carlo search algorithm to efficiently construct complex structures from electron density maps. Dr. James M . Gruschus is applying this method in his structure study of the peroxiredoxin complex. Peroxiredoxins (Prx) are one of several classes of proteins that reduce peroxides, reactive oxygen species produced as a by-product of cellular metabolism. For modeling the complex, the ATP-bound human Sulfiredoxin (hSrx) was docked to hyperoxidized Prx II using EMAP. In the docked structures with the Prx cysteine-sulfinic residue closest to the hSrx reactive cysteine, Asn186 of Prx II is in contact with the hSrx-bound ATP beta and gamma phosphate groups.[unreadable] [unreadable] 4. Xray Crystallography[unreadable] Dr. Parry has reported the structure of the class II major histocompatibility complex DRA, DRB3 in complex with an integrin peptide (PDB code 2Q6W). This is the first structure from the DRB3 gene locus. The structure models at high resolution the molecular basis of severe blood diseases associated with pregnancy, neonates, blood transfusion, stem cell and tissue transplant. A second structure is a complex with an altered ligand has been refined and awaits submission.